This document describes a technique for bioassaying histamine in the presence of prostaglandins using an Amberlite XAD-2 column. The column removes prostaglandins, rabbit aorta contracting substance, and possibly slow reacting substance of anaphylaxis, but allows histamine to pass through. Two banks of bioassay organs (rabbit aorta strip, rat stomach strip, guinea pig ileum) are used, with one bank above the column and one below. This allows accurate measurement of histamine levels in samples also containing prostaglandins and other substances that interfere with histamine bioassay. The method was tested using effluent from shocked, perfused guinea pig lungs and showed improved accuracy

1. Br. J. Pharmac. (1976), 56, 39-41
BIOASSAY OF HISTAMINE
IN THE PRESENCE OF PROSTAGLANDINS
R.J. GRYGLEWSKI & R. KORBUT
Department of Pharmacology, Copernicus Academy of Medicine, 16 Grzegorzecka, 31-531 Cracow, Poland
I An Amberlite XAD-2 column in a heated (37 0C) jacket was incorporated in between two banks of
bioassay organs superfused with Krebs bicarbonate solution in cascade. The column removed pro-
staglandins El, E2 and F2a and also rabbit aorta contracting substance (RCS) and possibly slow
reacting substance of anaphylaxis (SRS-A).
2 The column gave free passage to histamine in concentrations up to 3000 ng/ml. The method
described improves the accuracy of histamine bioassay in the presence of prostaglandins.
Introduction
The technique of Vane (1964) has been used for a gassed with 95% 02 and 5% CO2. Krebs solution
simultaneous bioassay of histamine, slow reacting (37°C) was dripped over the organs at a rate of
substance of anaphylaxis (SRS-A), prostaglandins and 2.5 ml/min, or perfused through the isolated lungs of
rabbit aorta contracting substance (RCS) in the sensitized guinea-pigs before reaching the organs
effluent from the shocked perfused lungs of sensitized (Piper & Vane, 1969). All substances were infused
guinea-pigs (Piper & Vane, 1969, 1971; Palmer, Piper directly over the organs at a rate of 0.2 ml/min except
& Vane, 1973; Piper, 1974). The cat terminal ileum for ovalbumin (1O mg), which was injected into the
(Ferreira, Ng & Vane, 1973) and the mepyramine- pulmonary artery. The combined antagonists
treated guinea-pig trachea were used to differentiate (Gilmore, Vane & Wyllie, 1968), minus mepyramine,
between histamine and SRS-A, and prostaglandins were infused directly over the tissues. The movements
and RCS were bioassayed by standard procedures of the assay organs (initial load 2-3 g) were recorded
(Ferreira & Vane, 1967; Piper & Vane, 1969). with Paton's auxotonic levers connected to Harvard
We have observed that the peak concentration of transducers (type 386) and a Watanabe multirecorder.
histamine released during anaphylactic shock from Amberlite XAD-2 (Keirse & Turnbull, 1973) or
perfused guinea-pig lungs, as indicated by the glass wool (in control experiments) were used to fill the
difference between the responses of the antazoline-free column (2 x 5 cm). The column was maintained at
and antazoline-treated guinea-pig ileum (1.08 jtg/ml, 37°C by a water jacket and placed between the two
s.e. + 0.21, n= 7) is much higher than that determined banks of the assay tissues (Figure 1). Before being
spectrofluorimetrically by the method of Hakanson & used the Amberlite column was washed twice with
R6nnberg (1974) (0.23 ,g/ml, s.e. ± 0.04, n= 7). One 100 ml portions of distilled water and then perfused
of the reasons for this discrepancy may be the with Krebs (2.5 ml/min) for 15 minutes.
presence of prostaglandins and RCS in the effluent. The following substances were used: prostaglandins
Here we describe a technique, which enables E1, E2 and F2a (Upjohn Co.), histamine
histamine to be bioassayed by the guinea-pig ileum in dihydrochloride (Polfa), antazoline methane-
the presence of relatively high concentrations of sulphonate (Polfa), mepyramine maleate (May and
prostaglandins. Baker) and Amberlite XAD-2 (BDH Chemicals Ltd).
Methods Results
Two banks of the assay organs, each consisting of a In our experiments the threshold concentrations of
rabbit aortic strip, a rat stomach strip and a guinea-pig prostaglandins E1 and E2 which contracted the rat
ileum, were superfused with Krebs bicarbonate stomach strip were 0.5-2 ng/ml, and those of pros-
solution of the following composition (mM): NaCl taglandin F2a were 2-10 ng/ml. The guinea-pig ileum
118.0, KCI 4.7, KH2PO4 1.2, MgSO4 7H2O 1.17, was usually contracted by histamine in concentrations
CaCl2 6H20 2.5, NaHCO3 25.0 and glucose 8.4, of 5-10 ng/ml, and also by prostaglandins El and E2

2. 40 R.J. GRYGLEWSKI & R. KORBUT
Perfused Lungs Lungs II at 10-150 ng/ml and also RCS from shocked guinea-
lungs 10 min pig lungs are reduced to undetectable levels during one
b 750OmV passage through XAD-2. Amberlite absorbs neither
histamine (10-3000 ng/ml) nor catecholamines
RbALJ (10-100 ng/ml). The guinea-pig ileum pretreated with
antazoline (0.5 jig/ml) or with mepyramine
(1.0 jg/ml) and situated beneath the XAD-2 column
RSS -J* was contracted neither by histamine nor by the
effluent from shocked lungs (5 experiments). It seems
likely therefore that SRS-A is also absorbed by
GPI XAD-2.
Using the above method we have found that the
Off On effluent from shocked lungs of sensitized guinea-pigs
XAD-2 contains histamine in the range 225-700 ng/ml
(470 ± 54 ng/ml, mean ± s.e., n = 10) together with a
RbA L _J prostaglandin-like substance (43.1 + 3.7 ng/ml pros-
taglandin E2 equivalents).
RSS
Discussion
b * *4 U * 4 *
E2 H E2 H E2H An Amberlite XAD-2 column removes pros-
mgmlr1minr1 30200 50 400 40400 taglandins, RCS and possibly SRS-A, but not
OVA OVA histamine from the effluent from the shocked lungs of
10mg 10mg
sensitized guinea-pigs, and therefore the guinea-pig
ileum situated distal to the column registers the actual
Figure 1 Isolated lungs of two sensitized guinea- amount of histamine in the effluent. A similar
pigs (lungs I and lungs 11) were perfused with Krebs absorbent column, but filled with aluminium oxide,
bicarbonate solution (2.5 ml/minute). The effluent has been proposed by us for the bioassay of pro-
was used to superfuse two banks of the assay tissues. staglandins in the presence of high concentrations of
Each consisted of a rabbit aortic strip (RbA), a rat
stomach strip (RSS), and a piece of guinea-pig ileum catecholamines (Korbut, 1975; Grodzifiska,
(GPI). All tissues were blocked with combined Panczenko & Gryglewski, 1975).
antagonists minus mepyramine (Gilmore et al., Differential assay of biologically active substances
1968). Calibration amounts (U) of prostaglandin E2 in a mixture is possible with organ cascades because
(E2) and histamine (H) were infused directly into the of the selective sensitivity of the assay organs towards
effluent at the top of the cascade. Antigen (ovalbumin individual hormones (Vane, 1964). This may be
10 mg) was injected into the pulmonary artery (OVA). further improved by administration of combined
During the perfusion of lungs 11 the XAD-2 column antagonists (Gilmore et al., 1968). The procedure
was placed between the two banks of tissues. The described here provides another way of facilitating the
difference in the responses of the guinea-pig ilea
above and below the column shows the influence of specific bioassay of hormones and autacoids in
the interfering substances on histamine bioassay by mixtures.
the upper guinea-pig ileum.
We thank the Trustees of The Welicome Trust (London) for
the generous grant of equipment, and Dr J. Pike, Upjohn
in concentrations of 10-20 ng/ml. As partially Co., Kalamazoo, U.SA. for kindly supplying us with pros-
exemplified in Figure 1, prostaglandins E1, E2 and F2a taglandins.
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3. HISTAMINE ASSAY IN THE PRESENCE OF PGs 41
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